Saxifraga L., the largest genus in the Saxifragaceae, consists of approximately 450 species and is distributed in temperate to alpine regions of Eurasia and North and South America. Among the 216 species found in China, mainly in Sichuan and Yunnan provinces and Xizang (Tibet) Autonomous Region, 139 are endemic (Pan et al., 2001). Saxifraga egregia Engl. is a perennial herb that is endemic to the Qinghai–Tibet Plateau and mainly inhabits forests, forest understories, and scrubs, with an elevation of 2000–4600 m a.s.l. (Pan et al., 2001). Saxifraga egregia and its ca. 30 close relatives are of great importance in the fields of systematics and phylogeography to extend our knowledge of the patterns and processes of speciation and intraspecies diversification in alpine regions. They are also excellent organisms for investigating biotic responses to climate change (DeChaine et al., 2013). Microsatellites have become one of the most popular molecular markers because of their high polymorphism levels and the relative ease of scoring, and they have been used in systematic and phylogeographic applications (Zane et al., 2002). In this study, we isolated polymorphic microsatellite loci of S. egregia to facilitate our further investigations of systematics and phylogeography.
METHODS AND RESULTS
Fifty S. egregia individuals from three populations (BM, DG, and CY) were sampled in Qinghai Province, Sichuan Province, and Xizang Autonomous Region (Appendix 1). Fresh leaves were collected and dried using silica gel.
Genomic DNA extraction, magnetic bead enrichment, and microsatellite-enriched library construction were performed according to published methods (Khan et al., 2014). Fragments from the microsatellite-enriched library were cloned into the pGEM-T Easy Vector (Promega Corporation, Madison, Wisconsin, USA), and then transfected into Trans5α Chemically Competent Cells (Trans-Gen, Beijing, China).
A total of 2520 positive colonies were successfully screened using PCR with primer-probes (AC)10/(AG)10. The amplified PCR products showed two or more bands on agarose gel electrophoresis (Skinner and Denoya, 1992). Of these, 320 randomly selected positive colonies were sequenced using an ABI 3730xl DNA sequencer (Applied Biosystems, Foster City, California, USA) according to the manufacturer's instructions at the Key Laboratory of Adaptation and Evolution of Plateau Biota, Chinese Academy of Sciences. SSR Hunter software for the analysis of simple sequence repeats (SSR) was used to detect 1200 microsatellite motifs (Li and Wan, 2005). A total of 112 primers were designed using online software Primer3 version 4.0.0 (Rozen and Skaletsky, 1999; http://primer3.ut.ee/), with the minimum and maximum primer annealing temperature changed to 58°C and 60°C, respectively, based on a total of 112 randomly selected microsatellite motifs.
Loci polymorphism in the 50 S. egregia individuals was assessed by PCR with designed primer pairs. PCR was performed in 20-µL reaction volumes containing 10–100 ng of template DNA, 1× PCR Buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 200 nM of each primer, and 1 unit of Taq DNA polymerase (TaKaRa Biotechnology Co., Dalian, China). The PCR cycling profile included an initial step of 94°C for 5 min; followed by 35 cycles of 94°C for 45 s, primer annealing temperature (Table 1) for 30 s, and 72°C for 30 s; with a final extension step at 72°C for 7 min. All PCR products were analyzed by capillary electrophoresis using the QIAxcel DNA high-resolution kit (1200) in the QIAxcel Advanced system (QIAGEN, Hilden, Germany). Biocalculator QIAxcel software was used for data analysis and generation of a virtual gel image.
Preliminary population genetic analyses, including the number of alleles (A), observed (Ho) and expected (He) heterozygosities, deviations from Hardy—Weinberg equilibrium (HWE), and linkage disequilibrium (LD) between all pairs of polymorphic loci, were calculated using GENEPOP version 4.2 (Raymond and Rousset, 1995; Rousset, 2008). Significance testing of the inbreeding coefficient (FIS) at all loci was performed using FSTAT 126.96.36.199 (Goudet, 2002). MICRO-CHECKER (van Oosterhout et al., 2004) was used to detect null allele frequencies (r) for all loci.
Characteristics of 12 polymorphic microsatellite loci in Saxifraga egregia.
Of the 112 primer pairs, 48 generated amplification products of expected sizes. Twelve of these displayed polymorphism, and their characteristics are shown in Table 1. Information on the 36 monomorphic primer pairs is listed in Appendix 2. Overall, A ranged from four to 14 per locus across 50 individuals (Table 2). Ho and He ranged from 0.421 to 1.000 and 0.622 to 0.939 per locus, respectively, which suggests that genetic diversity in this species is relatively high (Chen et al., 2009). This could be the result of interspecific hybridization between S. egregia and its closely related species with sympatric distribution ranges (e.g., S. diversifolia Wall. ex Ser.), considering their quite similar morphological features (Pan et al., 2001). Unfortunately, there is almost no research about the mating system of Saxifraga, and further study is needed before definite conclusions can be drawn. No linkage disequilibrium was detected in any pair of loci. Most loci (three, nine, and eight in populations DG, CY, and BM, respectively) showed a significant departure from HWE, consistent with the inbreeding coefficient. MICRO-CHECKER suggested that this may be affected by the presence of null alleles (Chapuis and Estoup, 2007).
Initial primer screening in Saxifraga egregia.a
- M. P. Chapuis , and A. Estoup . 2007. Microsatellite null alleles and estimation of population differentiation. Molecular Biology and Evolution 24: 621–631. Google Scholar
- F. Chen , A. Wang , K. Chen , D. Wan , and J. Liu . 2009. Genetic diversity and population structure of the endangered and medically important Rheum tanguticum (Polygonaceae) revealed by SSR markers. Biochemical Systematics and Ecology 37: 613–621. Google Scholar
- E. G. DeChaine , S. A. Anderson , J. M. McNew , and B. M. Wendling . 2013. On the evolutionary and biogeographic history of Saxifraga sect. Trachyphyllum (Gaud.) Koch (Saxifragaceae Juss.). PLoS One 8: e69814. Google Scholar
- G. Khan , F. Zhang , Q. Gao , X. Jiao , P. Fu , R. Xing , J. Zhang , and S. Chen . 2014. Isolation of 16 microsatellite markers for Spiraea alpina and S. mongolica (Rosaceae) of the Qinghai–Tibet Plateau. Applications in Plant Sciences 2: 1300059. Google Scholar
- Q. Li , and J. M. Wan . 2005. SSR Hunter: Development of a local searching software for SSR sites. Hereditas (Beijing) 27: 808–810. Google Scholar
- J. T. Pan , R. J. Gornall , and H. Ohba . 2001. Saxifraga L. In Z. Y. Wu and P. H. Raven [eds.]. Flora of China, vol. 8. Science Press, Beijing, China, and Missouri Botanical Garden Press, St. Louis, Missouri, USA. Google Scholar
- M. Raymond , and F. Rousset . 1995. GENEPOP (version 1.2): Population genetics software for exact tests and ecumenicism. Journal of Heredity 86: 248–249. Google Scholar
- F. Rousset 2008. GENEPOP'007: A complete re-implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources 8: 103–106. Google Scholar
- S. Rozen , and H. Skaletsky . 1999. Primer3 on the WWW for general users and for biologist programmers. In S. Misener and S. A. Krawetz [eds.]. Methods in molecular biology, vol. 132: Bioinformatics methods and protocols. 365–386. Humana Press, Totowa, New Jersey, USA. Google Scholar
- D. D. Skinner , and C. D. Denoya . 1992. A simple DNA polymerase chain reaction method to locate and define orientation of specific sequences in cloned bacterial genomic fragments. Microbios 75: 125–129. Google Scholar
- C. van Oosterhout , W. F. Hutchinson , D. P. M. Wills , and P. Shipley . 2004. MICRO-CHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4: 535–538. Google Scholar
- L. Zane , L. Bargelloni , and T. Patarnello . 2002. Strategies for microsatellite isolation: A review. Molecular Ecology 11: 1–16. Google Scholar
 This work was financially supported by the National Natural Science Foundation of China (grants no. 31270270, 31400322, and 31110103911), the International Scientific and Technological Cooperation Projects of Qinghai Province (no. 2014-HZ-812), and the West Light Foundation of the Chinese Academy of Sciences.